Recent studies suggest that CD4+ regulatory T (Treg) cells can suppress the CD4+ and CD8+ effector cell responses needed for optimal antitumor immunity. These observations may explain, at least in part, why attempts to elicit strong and durable antitumor responses with cancer vaccines (comprising MHC class I- restricted tumor antigens or peptides) have been largely unsuccessful. The goals of this project are to develop new strategies to shift the balance from CD4+ Treg cell-mediated suppressive immune response toward effective T helper immune response, thus generating long-lasting antitumor immunity for the ultimate tumor destruction. The underlying rationale is that tumor cells not only provide a suppressive environment by secreting interleukin (IL)-10 and TGF-beta, but also actively recruit and activate CD4+ Treg cells at local tumor sites by presenting tumor-associated or -specific ligands. These tumor antigen-specific CD4+ Treg cells, in turn, exert potent suppressive effects on effective T cell responses against cancer. Research in the applicant's laboratory has established several antigen-specific CD4+ Treg cell lines and clones from clinical samples taken from cancer patients. These unique cells, together with several newly developed technologies, should enable identification of important ligands for tumor-specific CD4+ Treg cells, permitting in turn more rigorous testing of a novel concept--that shifting the dynamic balance from suppressive CD4+ Treg cells to a milieu favoring T-helper-mediated antitumor responses may boost the effectiveness of cancer immunotherapy. To test our novel concept and hypothesis, we proposes three specific research aims: (1) use established CD4+ Treg cell lines/clones to identify genes encoding the ligands of these cells and then characterize the natural properties of the protein products;(2) dissect the immunosuppressive mechanisms and regulation of the CD4+ Treg cells to gain critical information needed for hypothesis testing in animal models;(3) use TCR transgenic mice and the B16 tumor model to modulate or reverse the suppressive function of CD4+ Treg cells in vivo. The strategies emerging from this 5-year proposal will be applied to different tumor antigens, tested in different animal models, to verify that a shift in the CD4+ Treg/effector cell balance is indeed conducive to more effective cancer immunotherapy. A positive outcome of these studies would open new opportunities for treating cancer patients and perhaps infectious and autoimmune diseases as well.